stained with anti-CD28-FITC, anti-CD4-PerCp Cy5.5, and
anti-Foxp3-allophycocyanin (all from eBioscience). Intracellular staining was
done after surface staining using a Foxp3 intracellular staining kit
(eBioscience). To determine total numbers of Foxp3+ Tregs,
the number of spleen cells was multiplied by the total percentage of
CD4+Foxp3+ cells detected by flow cytometry. Flow
cytometry was done using a Cyan ADP flow cytometer (Beckman Coulter) and data
analyzed using FlowJo (TreeStar Inc). For assessment of CD4+and CD8+ T cells in spleens of antibody treated mice or
separated T cell subsets, cells were stained with PE-anti-CD4 (RMA-4;
eBioscience or Biolegend) or PE anti-CD8 (53.6; eBioscience or Biolegend).
Samples were analyzed on a FACSCalibur (BD Bioscience) and data analyzed using
FlowJo.